1. Field of the Invention
This invention relates to the field of blood substitutes, more specifically, a method for producing an oxygen-carrying encapsulated hemoglobin material that is sterile filterable.
2. Description of the Background Art
Liposome encapsulated hemoglobin, also known as a synthetic red blood cell, has been under development as a blood substitute for over a decade. For example, liposome encapsulated hemoglobin formulations and methods for making them are known in the art: U.S. Pat. Nos. 4,776,991 and 4,911,929 (Farmer), 4,532,130 (Djordjevich), 4,133,874 (Miller), and 5,589,189 (Moynihan). All documents cited herein are hereby incorporated by reference thereto. Liposomes are closed hollow spheres formed by a bilayer membrane of phospholipids. As liposomes are formed, water soluble molecules, such as hemoglobin, can be incorporated into the aqueous interior, and lipophilic molecules may be incorporated into the lipid bilayer. Liposomes may be either multilamellar, like an onion with liquid separating many lipid bilayers, or unilamellar, with a single bilayer surrounding an aqueous center. Unilamellar vesicles generally have a size less than 0.5 microns (um). Smaller unilamellar vesicles can be formed by a variety of techniques, such as applying a force sufficient to reduce the size of the liposomes and/or produce smaller unilamellar vesicles. Such force can be produced by a variety of methods, including homogenization, sonication or extrusion (through filters) of multilamellar vesicles. These methods result in dispersions of unilamellar vesicles having diameters of up to 0.2 um.
One important characteristic of a regulatory approved blood substitute or parenteral product is that it be sterile. Terminal sterile filtration is preferred to aseptic processing for the generation of a sterile parenteral product, and has been found to be the most effective in terms of processing and liposome stability. The best method for terminal sterile filtration is the sequential filtration of a dispersion of liposomes through a 0.45 and 0.22 um filtration system, and liposomes larger than 0.2 um, or aggregation of smaller liposomes will obstruct and clog this filter system as well as the ultrafiltration system employed to remove unentrapped components. The Farmer patents disclose the small scale filtration of a liposome encapsulated hemoglobin formulation dispersed in a hyperosmotic buffered saline solution through a 0.22 um filter. Similarly, Djordjevich discloses a laboratory process for filtering liposome encapsulated hemoglobin dispersed in a saline solution through a 0.22 micron filter for purposes of sterilization.
An important aspect of developing a liposome formulation, whether containing hemoglobin or a desired reagent or drug, is to achieve a unimodal or controlled particle size distribution of unilamellar liposomes having a median size less than 0.2 um. Controlling a particle size distribution provides not only for a sterile filterable product but it also provides other numerous processing and pharmacological benefits such as longer circulation time.
During the processing of liposome encapsulated hemoglobin, hemoglobin enters the liposome as expected, but is also associated with the outer surface of the liposome as evidenced by electron micrographs, and uv-visible spectroscopy. The surface-bound hemoglobin has been shown to be responsible for aggregation of the liposomes. The liposome aggregation has been the focus of much manufacturing efforts as the aggregate size is not amenable to 0.22 or 0.45 um sterile filtration, an essential feature of a large scale manufacturing process required for FDA approval.
Passivation of the surface of the liposome with a serum protein is known in the art (see Moynihan, U.S. Pat. No. 5,589,189) and has been used in an attempt to produce sterile filterable liposome dispersions. However, all of the samples processed with methods in the art display heterogeneous size distributions and sizes which do not allow 0.22 um filtration.
Therefore, the need exists for a method of producing liposome dispersions, specifically, liposome encapsulated hemoglobin that renders particle size distributions which allow sterile filtration.